Polyethylene glycol (PEG) is a polymer that is commonly used as a covalent adduct to many biotherapeutic agents to increase their circulatory half life. By increasing the hydrodynamic radius of the molecule, PEG reduces loss due to glomerular filtration and leading 12 to decreased renal clearance. Kang et al. (2009) Expert Opin. Emerg. Drugs 14:363; Fishburn et al. (2008) J. Pharm. Sci. 97:4167. The PEG moiety on the therapeutic may vary in length (20 to 40 kD) and in branching.
PEG is also known to be ubiquitously present in food and cosmetic products to which human exposure is believed to elicit anti-PEG antibodies. Hershfield et al. (2014) Arthritis Res. Ther. 16:R63; Schellekens et al. (2013) Pharm. Res. 30:1729; Garay et al. (2012) Expert Opin. Drug Deliv. 9:1319. The PEG in such products is generally smaller and simpler in structure than PEG that is linked to a protein therapeutic.
Immunogenicity of biotherapeutics is known to influence their bioavailability, in vivo pharmacological potency, and immune complex mediated adverse effects. Gorovits et al. (2014) J. Immunol. Methods 408:1. Although PEG has been considered to be of low immunogenic risk due to its simple subunit repeat structure and low charge density, there have been several reports documenting both pre-existing anti-PEG antibodies in humans as well as therapeutic induced anti-PEG antibody response. Armstrong et al. (2007) Cancer 110:103; Hershfield et al. (2014) Arthritis Res. Ther. 16:R63; Ganson et al. (2006) Arthritis Res Ther. 8:R12. In some instances drug induced anti-PEG antibodies have been shown to increase the clearance of a PEGylated enzyme therapeutic. Armstrong et al. (2007) Cancer 110:103. Several groups have also raised both IgM and IgG antibodies against PEG in mice. Cheng et al. (2005) Bioconjug. Chem. 16(5):1225; Su et al. (2010) Bioconjug. Chem. 21:1264.
Most immunogenicity assays developed for a PEGylated therapeutic protein determine anti-drug antibody (ADA) response to the whole drug without discriminating between the specificity of the ADA towards the protein and PEG components of the therapeutic molecule. Some groups have addressed these questions by using PEG as a competitor in the ADA assay. Liu et al. (2011) J. Pharmacol. Toxicol. Methods 64:238; Hershfield et al. (2014) Arthritis Res. Ther. 16:R63. Others have used a separate bridging assay that uses an IgM anti-PEG positive control, but such an assay would only detect an IgM response. That most pre-existing anti-PEG antibodies might be IgM might make such an assay relevant. However, it is conceivable that antibodies induced by sustained chronic exposure to a PEGylated therapeutic, or food and cosmetic additives, might mature to an IgG isotype and would not be detected in an IgM bridging assay. Depending on the patient's immune status and the therapeutic indication, ADA of all isotypes specific to PEG may be relevant to drug safety in humans and therefore need to be monitored.
Our work has led to the realization that an ideal assay to detect such anti-PEG ADA would be (a) capable of detecting anti-PEG antibodies of wide range of affinities, with a broad specificity across all PEG sizes and shapes (b) a generic assay suitable for any PEGylated therapeutic drug regardless of the underlying protein portion of the drug and (c) and capable of identifying antibodies of all isotypes (IgM and IgG). See, e.g., Myler et al. (2015) Bioanalysis 7:1093. We also realized that the multiplicity of the epitopes on the PEG backbone might preclude IgG anti-PEG ADA detection in the traditional bridging assay format by virtue of intra-chain binding rather than inter-chain bridging between molecules. Binding of both arms of an IgG anti-PEG to the same PEG chain would preclude use of a bridging assay, and necessitate a direct assay format involving detection of the Fc region of the antibody. In such assays the Fc in positive controls and experimental samples will need to be of the same species. Such analytical requirements need a panel of well characterized anti-PEG reagents preferably with a human IgG Fc.
The need exists for improved reagents for analytical experiments to characterize PEGylated molecules, such as PEGylated therapeutic molecules, including but not limited to PEGylated proteins, antibodies, or biologically active fragments thereof. Such reagents would ideally allow for detection, and optionally quantification, of such PEGylated molecules or free PEG in biological samples or tissues in a variety of applications besides immunoassays. Such improved reagents would simplify experimental design, improve precision and enable detection of IgG antibodies.